scholarly journals Identification of a catalytically essential nucleophilic residue in sheep liver cytoplasmic aldehyde dehydrogenase

1991 ◽  
Vol 275 (1) ◽  
pp. 207-210 ◽  
Author(s):  
T M Kitson ◽  
J P Hill ◽  
G G Midwinter
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Tryptic Digestion ◽  
Sheep Liver ◽  
Unequivocal Identification ◽  
Peptide Fractionation

Sheep liver cytoplasmic aldehyde dehydrogenase was labelled by reaction with the substrate p-nitrophenyl di[14C]methylcarbamate. After tryptic digestion and peptide fractionation the labelled residue was identified as Cys-302. This is the first unequivocal identification of the essential enzymic nucleophile in the esterase activity of aldehyde dehydrogenase. By implication, Cys-302 is probably also the residue that is acylated by aldehyde substrates and the first residue that is modified by disulfiram.

scholarly journals Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase

1982 ◽  
Vol 205 (2) ◽  
pp. 443-448 ◽  
Author(s):  
F M Dickinson ◽  
G J Hart
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Metal Ion ◽  
Stopped Flow ◽  
Maximal Effect ◽  
Ion Concentrations ◽  
Sheep Liver ◽  
High Concentrations ◽  
Flow Experiments ◽  
Active Centres

Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.

scholarly journals Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase

1978 ◽  
Vol 175 (1) ◽  
pp. 83-90 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Enzyme Activity ◽  
Linear Relationship ◽  
Dehydrogenase Activity ◽  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Competitive Inhibitor ◽  
Enzyme Molecule ◽  
Covalent Interaction ◽  
Sheep Liver ◽  
Non Linear

The effect of disulfiram, [1-14C]disulfiram and some other thiol reagents on the activity of cytoplasmic aldehyde dehydrogenase from sheep liver was studied. The results are consistent with a rapid covalent interaction between disulfiram and the enzyme, and inconsistent with the notion that disulfiram is a reversible competitive inhibitor of cytoplasmic aldehyde dehydrogenase. There is a non-linear relationship between loss of about 90% of the enzyme activity and amount of disulfiram added; possible reasons for this are discussed. The remaining approx. 10% of activity is relatively insensitive to disulfiram. It is found that modification of only a small number of groups (one to two) per tetrameric enzyme molecule is responsible for the observed loss of activity. The dehydrogenase activity of the enzyme is affected more severely by disulfiram than is the esterase activity. Negatively charged thiol reagents have little or no effect on cytoplasmic aldehyde dehydrogenase. 2,2′-Dithiodipyridine is an activator of the enzyme.

scholarly journals Effect of some thiocarbamate compounds on aldehyde dehydrogenase and implications for the disulfiram ethanol reaction

1991 ◽  
Vol 278 (1) ◽  
pp. 189-192 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Dehydrogenase Activity ◽  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Negligible Effect ◽  
Weak Inhibitor ◽  
Low Concentration ◽  
The Third ◽  
Sheep Liver ◽  
Physiological Phenomenon

The effects of S-methyl diethyldithiocarbamate, S-methyl diethylmonothiocarbamate and bis(diethylcarbamoyl) disulphide on sheep liver cytoplasmic aldehyde dehydrogenase were investigated in vitro. The first compound has negligible effect. The second one is a weak inhibitor of the esterase activity of the enzyme and a weaker inhibitor of the dehydrogenase activity. A very low concentration of the third compound, however, acts as a potent inactivator of aldehyde dehydrogenase, similar in this respect to disulfiram, although somewhat slower to react. The possible involvement of these compounds in the physiological phenomenon known as the disulfiram ethanol reaction is discussed.

scholarly journals Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents

1989 ◽  
Vol 261 (1) ◽  
pp. 281-284 ◽  
Author(s):  
K M Loomes ◽  
T M Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Related Compound ◽  
Physiological Responses ◽  
Enzymic Activity ◽  
Cysteine Residues ◽  
Mitochondrial Enzyme ◽  
Step Process ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver

Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2′-dithiodipyridine and 4,4′-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4′-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2′-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5′-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.

scholarly journals Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase

1985 ◽  
Vol 225 (1) ◽  
pp. 159-165 ◽  
Author(s):  
F M Dickinson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Dissociation Reaction ◽  
Quenching Effect ◽  
Sheep Liver ◽  
Association Reaction ◽  
Hydrolysis Of

The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.

scholarly journals The activation of aldehyde dehydrogenase by diethylstilboestrol and 2,2′-dithiodipyridine

1982 ◽  
Vol 207 (1) ◽  
pp. 81-89 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Enzyme Activity ◽  
Aldehyde Dehydrogenase ◽  
Human Liver ◽  
Common Property ◽  
Rabbit Liver ◽  
Aldehyde Dehydrogenases ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver ◽  
Low Concentrations ◽  
Liver Aldehyde Dehydrogenase

1. The activation of sheep liver cytoplasmic aldehyde dehydrogenase by diethylstilboestrol and by 2,2′-dithiodipyridine is described. The effects of the two modifiers are very similar with respect to variation with acetaldehyde concentration, pH and temperature. Thus the degree of activation is maximal when the enzyme is assayed at approx. 1 mM-acetaldehyde, is greater at 25 degrees C than at 37 degrees C, and is greater at pH 7.4 than at pH 9.75. With low concentrations of acetaldehyde both modifiers decrease the enzyme activity. 2. Diethylstilboestrol affects the sheep liver cytoplasmic enzyme in a very similar way to that previously described for a rabbit liver cytoplasmic enzyme. Preliminary experiments show that the same is true for a preparation of human liver aldehyde dehydrogenase. It is proposed that sensitivity to diethylstilboestrol (and steroids) is a common property of all mammalian cytoplasmic aldehyde dehydrogenases.

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